Treating conditions caused by abnormal growth of pathogens in body cavities

ABSTRACT

A cooling device and method inhibit the infectious growth of pathogens in a body cavity, primarily to cure VulvoVaginal Candidiasis (VVC) and Bacterial Vaginosis (BV) in the vagina, and to reduce biofilms. The device has a shell having an internal chamber containing a freezable or cooling filler that reduces the body cavity temperature. The device may be comprised of an optional coating compound (agent) covering at least a portion of an outer surface of the shell and an optional stainless steel sleeve that covers the shell or optional stainless steel particles that are impregnated into the shell material to enhance both thermal conductance and cooling efficacy. Pathogenic biofilms dissolve and bacteria colony counts decrease at lowered temperatures in body cavities, and infectious, temperature sensitive organisms (fungi, yeast, bacteria, viruses, parasites, and protozoans) become dormant, change morphology, or cease growing under the same lowered body cavity temperatures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/059,843, filed on22 Oct. 2013, which is a continuation-in-part of, and claims the benefitof priority from, U.S. Ser. No. 13/637,496, filed on 26 Sep. 2012, whichin turn is a national stage entry of PCT/US11/30052, which was filed on25 Mar. 2011, which in turn is a non-provisional designating the US ofprovisional patent application Ser. No. 61/318,338, filed on 28 Mar.2010, which is now expired. Each of the cited applications isincorporated by reference as if fully recited herein.

TECHNICAL FIELD

The present invention relates to a device and method for treatingconditions in a body cavity caused by an overgrowth of normal flora,including Candida yeast species and certain bacteria, and/or thepresence of pathogens, such as ‘filamentous’ Candida yeast species,viruses, bacteria and protozoans. The invention is a cooling ortemperature reducing treatment device to cure vaginal infections,including VulvoVaginal Candidiasis (VVC), Bacterial Vaginosis (BV), andto reduce biofilms.

BACKGROUND

Vaginitis is a non-specific term describing symptoms such as irritationand/or inflammation of the vagina that affects millions of women eachyear. Vaginal infections produce a variety of symptoms, such asdischarge, itching, burning, pain, swelling, odor, painful urination orbleeding. Infectious forms of vaginitis can be caused by bacteria,viruses, protozoa, parasites, or an overgrowth of ‘filamentous’ (hyphaland pseudohyphal) yeast, primarily the Candida species. The two mostcommon types vaginal infections are VulvoVaginal Candidiasis (VVC), alsoknown as yeast vaginitis, and Bacterial Vaginosis (BV). Yeast (primarilyCandida albicans) are dimorphic fungi and are normal inhabitants of skinand body cavities. Candida species can become a problem or pathogenicwhen they become virulent under the influence of heat, a requiredcondition for the pathogenesis of VVC. Other risk factors for thedevelopment of VVC besides heat and moisture are sunbathing, swimming,exercise, sexual intercourse, oral sex, tight fitting pants, panty hose,non-cotton crotched underwear, and thongs. Antibiotics, oral steroids,pregnancy, immunosuppressed states, diabetes mellitus, urinaryincontinence, and some medications (oral contraceptives) can predisposea woman to recurrent VVC. Beside nutrient restriction, pH changes, CO2,and serum, the requirement for an elevated temperature is essential forthe yeast to change size and shape by morphing into their ‘filamentous’forms (hyphal and pseudohyphal). These ‘filamentous’ forms areresponsible for the invasive disease state known as VVC. Candidaalbicans (C. albicans) and other species may form biofilms as a resultof VVC which results in the persistence of the condition and resistanceto treatment. The mechanism by which C. albicans are able to changetheir morphology is due to the increased production of HSP 90 (heatshock proteins by C. albicans and other species with similar proteins)which occurs as a direct result of elevated temperatures. This ‘heat’requirement for yeast to change into their virulent, filamentous formshas remained enigmatic.

Bacterial vaginosis (BV) is the most prevalent cause of infectiousvaginitis accounting for about 40-45% of all cases. It is distinguishedby a malodorous vaginal discharge that is adherent to the vaginal mucosaof body cavities. While Gardnerella vaginalis is associated with thecondition, BV is thought to be a synergistic, polymicrobial infection ofpathogens, primarily anaerobic, such as Atopium vaginae, mobiluncus,bacteroides, ureoplasma, mycoplasma, fusobacterium, among many others.As these pathogens and normal flora multiply, forming a biofilm, theyelaborate chemicals and waste products known to elevate the pH andproduce the characteristic ‘amine’ malodorous discharge. It is thisbiofilm that allows the bacteria of BV to remain resistant to antibiotictreatment and to frequent recurrences. Biofilms (‘Surface Slimes’) areadherent communities of microorganisms, held together by a polymericmatrix of polysaccharides, proteins, and nucleic acids. Biofilms andtheir biologic components such as bacteria, yeast, virus, and theirmetabolic waste products are electrostatically negatively charged.Current studies are underway to find enzymatic or electromagneticdissolution, so that lactic acid and hydrogen peroxide (H₂0₂) can berestored via their production by the normal bacterial flora, known aslactobacillus. Lactobacillus crispatus predominates in an healthyvagina. The current device will introduce temperature reduction to thetissue and adherent biofilm, to disrupt the biofilm with the optionaladdition of a coating compound such as pH normalizing or acidifyingagent to help normalize the vaginal pH. The elevation in pH from BVprobably occurs at a much later stage and is one of the reasons thatvaginal applications hydrogen peroxide (H2O2) and lactic acid have lesseffect on the persistence of the biofilm, and account for recurrences ofBV. Results from research studies show that biofilms exist ‘in vitro’ attemperatures of 37° C., making biofilms also temperature sensitive,especially at lowered tissue temperatures. Biofilms start to break downat less than 30 C and higher oxygen levels. Therefore, the optionaladdition of hydrogen peroxide to a cooling device inserted into a bodycavity will release oxygen and hydrogen ions that will increase theoxygen content of the vagina which causes the self-destruction ofbiofilms, will help restore the pH balance (acidification via H+ ionrelease), and encourage the re-colonization of lactobacilli species inthe vaginal epithelium, since this species does not live in biofilms,nor is it temperature sensitive. Lactobacilli produce hydrogen peroxideand lactic acid to keep the vaginal at a normal pH of about 3.5-4.5.Thus, the tissue cooling effect of this intra-vaginal device describedin the invention, will simultaneously disrupt biofilms, allow easierpenetration of any necessary chemicals or agents, and allow the normalflora of lactobacillus species to grow normally and restore the vaginato its normal pH.

The bacteria of BV are both anaerobic and facultative anaerobic and growbest under conditions with little or no oxygen. Fortunately,lactobacilli are facultative anaerobes and tolerate both conditions, butflourish best in the presence of oxygen.

Biofilms on the skin and in body cavities resist removal by conventionalsoap and water. A mechanism that could prove essential in helping withthe treatment and cure of these conditions is that the pathogens'biofilms may be disrupted simply by the mechanical separation of thevaginal (body cavity) walls, the lowering of the temperature of thevaginal tissue, or by applying an agent with an electrostatic positivecharge to cause adherence of the negatively charged exopolysaccharidebiofilms. For instance, polylysine, a cationic (positively charged)polypeptide used as a food preservative, can adhere to a cell's surface,causing clumping and cessation of protein synthesis, thus preventingspoilage via biofilm formation. BV is dependent on the biofilm fordisease persistence and low cure rates of approximately 80%, andbiofilms in VVC are less well understood as far as pathogenesis andpersistence, but may be a factor in more advanced cases or in personswith underlying conditions such as diabetes, pregnancy, orimmune-suppressed states.

Other causes of vaginitis may include vulvodynia and vestibulitis,conditions suspected to be caused by repetitive treatment withantifungal creams and repetitive antigenic exposure to Candida yeastspecies. This infectious destruction of tissue may result in poorhealing due to repetitive and over active immune responses, possiblycausing permanent ‘neuralgias’ or autoimmune ‘self’ destruction oftissue with resultant scarring, recalcitrant pain and burning. Sincethese conditions (vestibulitis and vulvodynia) are incurable and havepoor response to therapies, the present invention has the potential toprevent the need for repetitive chemical anti-fungal drug exposures,thus preventing the occurrence of these conditions in the first place.

There are over eighty-one varieties of yeast, three of which are Candidaalbicans, Candida tropicalis, Candida glabrata, while C. parapsilosis,C. kefyr, C. krusei are considered less common causes of VVC. For themost common species, Candida albicans accounts for 85-90% of cases ofVVC, and the over-the-counter remedies now available will treat and curethis type of yeast only 80% of the time. Current treatment for C.tropicalis, C. glabrata, and the others are by physician prescribedantifungal creams only. All current over-the-counter antifungal creamstreat only one form of VVC (Candida albicans), and take days, not hoursto be effective.

The current treatment systems use medicine/drugs (either oral ortopical) to treat VVC and BV. Likewise, there are no over-the-countertreatments for other types of pathogens in the vagina like trichomonas,gonorrhea, chlamydia, herpes, gardnerella (known to be a factor in BV)or any other, yet to be identified pathogen. The current therapiesrequire the use of anti-fungal or anti-microbial creams/semisolids ororal tablets. When the drug melts in the vagina, it spills out onto theperineum and causes more itching, burning, and pain, exacerbating thesymptoms associated with VVC and BV. These emulsions contain manysynthetic chemicals and preservatives, which are known to be caustic andallergenic to human skin and mucous membranes. These chemicals, mostnotably methyl parabens, propylene glycols, cetyl alcohols, sodiumlauryl sulfates are caustic to the skin, yet are used routinely in thecurrent antifungal medicines and other therapies (which do nothing morethan treat the itch symptom). Most clinicians believe there is anunderreporting of these skin reactions, thought to be a result of theover-the-counter manner in which the condition is treated, and due tothe fact that the toxic skin reactions mimic the symptoms of VVC, forwhich a woman seeks relief in the first place. Another problem with OTCand prescription anti-fungal medications is that their mechanism ofaction (destruction of the yeast cell membrane/wall) results in therelease of the cell's toxic contents (enzymes). These released enzymesresult in tissue destruction via the mechanism by which Candida are ableto penetrate tissue and invade the host. This increases the intensity ofthe symptoms by inflaming the already diseased tissue, prolonging thesymptoms for days, before any symptomatic relief is experienced.

Because the woman is confused by the claims of over-the-counter (OTC)medications due to the worsening of her symptoms, she is frustrated asher condition worsens until she either buys more irritating creams orvisits her doctor. With regard to BV, there are no OTC remedies for thiscondition, yet douche manufacturers routinely advocate douching as aneffective remedy for vaginal odor, inferring that their products treatthis condition. However, BV is known to be more common in women whodouche, suggesting that douching chemicals are the actual cause orincrease the risk of developing BV, rather than the solution. Allobstetricians/gynecologists advise against douching for many welldocumented scientific reasons (primarily due to increasing the risk ofBV, which is a known risk factor for both premature rupture of membranesand preterm births, as well as sexually transmitted diseases), yet theproducts persist, and are responsible for the recurring nature of thecondition. Furthermore, the only treatment options for BV are eithersystemic (oral) or topical drugs such as Metronidazole and Clindamycin,among others, in the same category of medications. The toxicities forthese two medications are noteworthy, with many side effects anddrug-drug interactions, as well as possible teratogenicity in pregnancy.These medications are available by prescription only. The inventiondescribed herein provides a safe, non-chemical solution for both VVC andBV to help reduce the risk of acquiring vestibulitis/vulvodynia ordelivering a premature infant. The cooling device is safer, worksfaster, and more effectively than chemical alternatives. A non-chemicaloption that has immediate symptomatic relief and a more effective remedyagainst all temperature sensitive pathogens and species of Candida, andthe potential to have an impact on BV via reduction in biofilms andbacterial counts, is the basis for this invention.

Existing medicinal creams add moisture and substances that promotebiofilm formation in the vagina, which is counterproductive to curingboth VVC and BV. Yeast can survive many environmental conditions, butunder normal pH and carbon dioxide levels, additional heat is necessaryfor yeast to change in size and shape (morphology), grow and multiply toinvade tissue, resulting in tissue destruction, enzyme release, andescape from immune cells causing the condition known as VVC. Undernormal body temperatures though, the mass destruction of lactobacillivia antibiotics, can result in a flare of VVC as a result of altered(increased pH). Candida albicans, which accounts for about 80-95% of VVCoutbreaks, can only be grown in a laboratory setting on appropriategrowth media at a minimum temperature of 36° C., which is 96.8° F.

Although current drugs do cure VVC infections about 80% of the time,they are not without significant side effects. Oral antifungal medicinealso poses significant risks, such as drug interactions resulting inanaphylactic shock or even death. Oral antifungals (fluconazole) used totreat VVC can be compared to using a “bazooka to shoot a mouse”; and,fluconazole only treats one species of yeast, Candida albicans.

Additional problems exist with currently available treatments forvaginitis caused by VVC. Current medications, while partially effective,may take 3-7 days to provide relief from itching, burning, swelling, andpain. In addition, both oral and topical drugs may adversely interactwith other medications, such as antihistamines, antidepressants, asthmamedications and the like. Life threatening anaphylaxis can result fromoral systemic medications. Some topical medications can cause toxic skinreactions. Furthermore, the success rate of existing treatment methodsrange from 60-80% because a chosen drug may not be effective against allof the strains of Candida and due to anti-fungal drug resistance, anincreasingly common problem due to the over-use or inappropriate use ofboth topical and oral anti-fungal medications. A need exists for atopical, non-chemical, method of treating both the symptoms and causesof VVC and BV infections within body cavities that is immediate,non-invasive, and effective against all yeast species, the bacterialovergrowth, and biofilms associated with BV, VVC, and other conditions.

Pregnancy is another predisposing condition whereby VVC and BV can causesignificant health risks to the mother and the baby such as in preterm,premature rupture of membranes. No currently available medications havebeen studied in pregnancy for these two conditions, and as such, areplaced in Category ‘C’, meaning unknown risk. A need exists forinhibiting pathologic fungal, microbial, viral, protozoan, and parasiticgrowth in a body cavity for both pregnant and non-pregnant women.

SUMMARY

The invention in an embodiment is a cooling device to inhibit thepathogenic or infectious growth of an organism and to disrupt a biofilmin a body cavity comprising a shell having an internal chambercontaining a freezable or cooling filler, an optional coating compoundcovering at least a portion of the outer surface of the shell, orimpregnated within the material comprising the shell, and an optionalstainless steel (SS) sleeve. The shell will be comprised of athermoplastic polymer or suitable medical grade material and optional SSparticles impregnated into the substance of the shell. When the deviceis inserted into the body cavity (vagina), the frozen or cooling fillerabsorbs body heat thus cooling the adjacent tissue in gradual manner todisrupt a biofilm and force any temperature sensitive pathogens intodormancy (quiescence) via reversal of morphology, via a ‘cold shockresponse’, inhibition of the heat shock response, or via some ‘other’yet to be described molecular or biochemical mechanism.

In an embodiment, the coating compound comprises a gel carrier and anagent. The gel carrier comprising a hydrogel having covalently linkedbiopolymer chains. The hydrogel swells upon contact with fluid causingdegradation of the gel carrier such that the agent is released from thecoating compound in a controlled manner and contacts the tissue wall ofthe body cavity. The agent is one, or a combination of, a pHnormalizing, pH acidifying, anti-microbial, anti-fungal, anti-parasitic,anti-protozoan, herbal, hormonal, steroidal, non-steroidal, oxygenreleasing (H2O2), anti-inflammatory, anti-oxidant, antiseptic,electrostatically charged, or probiotic substance, chemical, ingredient,or material. The gel carrier and agent may be pre-applied or mixed andapplied to the shell prior to insertion into the body cavity. In anembodiment, the compound is inserted separately from the device,immediately prior to or after insertion of the device into the bodycavity such that the device acts both to retain the compound in the bodycavity for an undetermined duration of time to take effect and causetemperature reduction in the cavity. The coating compound may comprise agel-forming solution or hardener to produce the coating. The coatingcompound may be liquid, particulate or powder, may be impregnated intothe shell material either during or subsequent to the manufacturingprocess. The coating compound may actually dissolve and be released fromthe shell surface upon contact with the tissue.

When the device is inserted into the body cavity, the freezable orcooling filler cools the body cavity via passive thermal conductance ortransference of heat in order to disrupt the biofilm, allowing theoptional coating compound (agent) better access to the pathogen(s) andto facilitate the disruption and breakdown of the biofilm. In theembodiment, the shell comprises SS particles to prolong the coolingeffect and to facilitate the conductance of heat away from the bodycavity tissues.

In an embodiment, the shell is enclosed inside a SS sleeve with thecoating compound applied to the exterior of that SS sleeve. The gelcarrier is comprised of a hydrogel having a network of covalently linkedbiopolymer chains. The hydrogel swells upon fluid contact causingdegradation of the gel carrier such that the agent is released from thecoating compound in a controlled release manner and contacts the tissuewalls of the body cavity, and the SS provides a longer, more steadyduration of cooling that results in biofilm disruption and pathogengrowth suppression, allowing more effective ‘agent’ access to thepathogens.

To use the device in order to inhibit the growth or reverse Candidaspecies morphology and to disrupt biofilms created by organisms in abody cavity, a user first cools or freezes the device, or allows for amixture of chemicals to create an endothermic reaction, which has anoptional SS sleeve covering the shell, which has an internal chambercontaining a cooling or freezable filler. The user then applies anoptional coating compound and covers at least a portion of the outershell of the cooling device or SS sleeve surface with the compound andinserts the device (with or without the SS sleeve) into the body cavity.After the device reaches approximately the same temperature as the bodycavity, the user removes the device from the body cavity and repeats theprocess until the infectious conditions have abated or until sufficientresearch is generated to determine the appropriate duration of use anddosage intervals.

The present invention is a cooling device, with an optional compounddelivery or retention function for body cavities to force Candidaspecies and other potential temperature sensitive pathogens into thedormant, non-disease state, to reduce an overgrowth situation, and todisrupt biofilms. Cooling of a body cavity to temperatures of less than30 C causes the disintegration of biofilms which are contributors to thedisease state of VVC, BV, and other pathological conditions of the bodycavity. The device is useful to treat problems such as VVC, an infectionof the vagina caused by the filamentous growth of Candida species,chemical dermatitis of the vagina and vulva, BV, an overgrowth of gramnegative, anaerobic bacteria (previously defined as BV), and anyassociated biofilm mediated conditions. The device may have similarutility for other temperature sensitive pathogens of the vagina, such astrichomonas and herpes, or for other body cavities. The presentinvention inhibits or reverses pathological ‘filamentous’ fungal(Candida species), may prevent or treat other microbial infections suchas BV, parasitic, protozoan, and viral infections while reducing biofilmformation simultaneously. The present invention's coating compound maybe pH neutralizing, pH acidifying, may contain inactive ingredients, maycontain anti-inflammatory such as aloe, anti-oxidant compounds,hormones, steroids, non-steroidal agents, herbs, anti-septics such asH2O2, lactic acid, anti-microbial, anti-viral, anti-parasitic,anti-fungal, anti-protozoan, electromagnetic, natural stabilizing agentsor artificial preservatives, oils, vitamins, or minerals as componentsof the coating compound housed in a gel, emulsion, powder, particles,creams, or emulsions or be impregnated into the shell of the device.

The invention addresses the optional application of a coating compound,medication, or topical chemical as described above, which aids in curingthe infection the body cavity, such that in conjunction with the coolingcomponent which disrupts biofilms and reverses infectious morphologies,helps the coating compound to be more effective, at lower dosages andwith less duration of exposure, allowing the normal host mechanisms toheal and repair the tissue faster, and to reduce the severity of thesymptoms, such as itching, burning, swelling, and pain. The normalinhabitants of the vagina (lactobacillus) can repopulate more quickly,ensuring a normal pH and healthy ecosystem of the body cavity.

The device, comprising a shell, with or without a removal means (stringor loop, for example) whereas the expulsion of the device can beaccomplished by a Valsalva maneuver or ‘bearing down’ as is the casewith normal ambulation, coughing, or lifting. The shell, with aninternal chamber filled with a freezable or cooling filler, and anoptional coating compound for the shell, may also be covered by a SSsleeve whereby the hollow sleeve chamber is filled with ambient airuntil it is placed over the shell of the device. The SS sleeve would beimportant to facilitate a prolonged cooling effect (enhanced thermalconductance), thus improving the overall effectiveness in curing theinfection and reducing biofilms in body cavities. In such case of a SSsleeve, the optional coating compound would cover the exterior surfaceof the SS sleeve.

When the shell with the cooled or frozen filler is inserted into a bodycavity, the device (invention) cools the body cavity to a temperaturethat causes the filamentous forms of Candida species to reverse back totheir yeast or bud shape, which is the dormant, non-disease formingstate and disrupts the biofilms which are associated with VVC and BV, aswell as biofilms associated with douching and other chemicals used infeminine hygiene products and soaps. This allows the non-temperaturedependent lactobacillus species to recolonize the vagina to normalizethe pH and allow the healing process to take place naturally. The filleris not in direct contact with the body cavity walls, but is separated bythe shell's wall thickness which is important with respect to the rateof transference of heat. The material of the filler is any cooling orfreezable substance, including but not limited to a liquid such assaline, distilled or bacteriostatic water, a gas, a solid or semi-solid,or a gel or hydrogel, or via the mixing of two or more substances tocause an endothermic reaction which has heat absorptive and coolingeffects on a body cavity.

The shell's chamber, which contains the filler, may comprise two or morecompartments separated by breakable walls. Each compartment is filledwith a substance such that when the wall is disrupted or broken, thesubstances combine to create a cooling effect via the endothermicreaction. This mixed substances, now the filler once used, can bere-cooled or frozen for re-use inside the shell and inserted into thebody cavity to reduce the tissue temperature on another occasion. Theremoval means is any mechanism or attachment to the shell such as astring, cord, loop, stalk, knob, button, ball, and the like, that whengrasped and pulled, will result in complete removal from the bodycavity. Or, in the case of no such attachment, removal will beaccomplished by simple Valsalva, coughing, or bearing down.

In the embodiment, an optional external (to the body cavity) coolingcomponent or extension may be attached to the shell after insertion, beintrinsic to the shell's design, or be a completely separate apparatusin order to apply cooling or cause heat transfer from the contiguousvulvar, clitoral, and perinea! tissue which also harbors the infectiouscondition such as Candida or other pathogenic organisms of theseregions. The external cooling component is comprised of a shell with afreezable or cooling filler inside an internal chamber as described inabove.

In an embodiment, the invention treats infectious diseases caused bypathogens such as Candida species (VVC), fungus, anaerobic andfacultative bacteria (BV) and their associated biofilms and thosebiofilms that may be a result of chemicals used in douching, femininehygiene products, soaps, body washes, and ingredients found inmedications or products used in body cavities or surfaces. When theshell with the cooling, freezable, or frozen filler is inserted into abody cavity, the filler gradually warms or thaws via passive thermalconductance such that the body cavity's tissue temperature is loweredwhich inhibits further virulent growth and reverses Candida species'morphology to the dormant, non-disease causing morphology, reducesbiofilms, allows for the optional addition of a coating compound toenhance the device's (invention's) efficacy and may serve as a substanceretention or delivery device in order for that compound to exertadditional therapeutic effect, at lower dosages, with reduced durationof exposure, and with reduced side effects or toxicities. The inventionis an optional substance retention device, whereby it limits thespillage of any compounds onto the contiguous tissues (perineum, vulva,clitoris) where the nerve supply is abundant thus resulting in thepainful and bothersome side effects associated with many compounds andchemicals. The optional SS sleeve to cover the invention's shellenhances thermal conductance, duration of cooling effect, and efficacy.

As used herein, “approximately” means within plus or minus 25% of theterm it qualifies. The term “about” means between ½ and 2 times the termit qualifies.

The compositions and methods of the present invention can comprise,consist of, or consist essentially of the essential elements andlimitations of the invention described herein, as well as any additionalor optional ingredients, components, or limitations described herein orotherwise useful in compositions and methods of the general type asdescribed herein.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range or to be limited to the exact conversion to adifferent measuring system, such, but not limited to, as between inchesand millimeters.

All references to singular characteristics or limitations of the presentinvention shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

Terms such as “top” “bottom” “right” “left” “above” “under” “side”“front” and “back” and the like, are words of convenience and are not tobe construed as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a see-through view of an embodiment of the invention showingthe chamber 12 for the filler 20.

FIG. 2 is a perspective view of an embodiment of the invention.

FIG. 3 is a diagrammatic side view of the coating compound of theinvention showing the polymer and the agent.

FIG. 3a is a diagrammatic side view of the coating compound beginning tobreak down and release agent.

FIG. 4 is a diagrammatic side view of further breakdown of the coatingcompound and the continued release of the agent.

DETAILED DESCRIPTION

Reference will now be made in detail to the exemplary embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings.

In accordance with an embodiment of the present invention as shown inFIG. 1, the device is comprised a shell 10, a filler 20 and an optionalcoating compound 40. The shell of the device, once cooled or frozen, isoptionally housed in a stainless steel sleeve (not shown). The devicemay include removal means 30 or 31. The shell is preferably made fromsilicone, medical grade thermoplastic polymers, or stainless steel (SS)and comprises an external wall 11, enclosing a chamber 12 that is sealedwith a plug 13. In the case of heat sealing after filling the chamber,there is no plug or cap and the device is a single unit. The chamber mayhave one or more compartments 14, 15. The compartments are formed withbreakable wall(s) 16 between or among them. The filler 20 is a non-toxicsubstance, such as but not limited to, saline, distilled water, a gel,or gas, and the like. The filler may comprise several components thatare housed apart in the compartments until use. In an embodiment, theremoval means 30 (if needed) is a string or cord firmly attached to theshell or to 31.

As shown in FIG. 3, the coating compound 40 comprises a gel carrier 42and an agent 41. The agent is a pH normalizing or acidifying agent,anti-microbial, anti-fungal, steroidal, non-steroidal, hormonal,anti-oxidant, anti-inflammatory, herbal, ati-septic, electromagnetic,oxygen releasing, or probiotic, or combination thereof. The compound isapplied to the shell prior to insertion or inserted separately by anapplicator prior to the device's body cavity placement. In the case ofelectromagnetic coatings, they may be applied during the manufacturingprocess, or impregnated into the shell material. Alternatively, thecoating compound is applied to the external surface of the SS sleeve(not shown).

The compound comprises a gel carrier 42 that is relatively soft. In anembodiment, the invention uses a gel-forming solution with a hardener toproduce the coating. The aqueous gel is relatively soft so that theaqueous gel does not adversely affect the device. In an embodiment, thecoating covers all of the device. Alternately, only portions of theexternal wall 11 are coated (with either a coating compound or stainlesssteel sleeve or particles).

The gel carrier is a chemical hydrogel having a network of biopolymerchains 43 covalently linked at strategic connection sites. The chemicalhydrogel is created by using crosslinkers 44 to covalently bridge thebiopolymers at specific selected sites, typically by reaction with smallmolecules. Upon contact with water, the hydrogel hydrates and swellsuntil an equilibrium state is reached, which depends on the extent ofthe crosslinking. The swelling process is governed first by waterbinding at the hydrophilic sites of the biopolymers, followed by theentrapment in the gel network.

As shown in FIG. 3a , diffusion of the agent occurs predominantlythrough the void spaces between the polymer chains. Addition ofplasticizer 45 (FIG. 3, both type and concentration) influences releaseof the agent 41. Plasticizers reduce polymer-polymer chain secondarybonding and provide mobility for the agent. Plasticizer leach out of thepolymer results in pore formation 46 for release of a portion of theagent 41. Subsequent release of agent is based on diffusion through thepolymer. Polyurethane, poly(ethylene vinyl acetate), andpolydimethylsiloxane are polymers used in the invention. Biodegradablepolymers, the polyesters, which consist mainly of poly(caprolactone),poly(lactic acid), poly(glycolic acid) and copolymers of lactic andglycolic acids are used in the invention. As depicted in FIG. 3a , agent41 is initially diffused by eroding of the compound. Polyanhydrides andpolyorthoesters degrade only at the surface of the polymer, resulting ina release rate that is proportional to the surface area of the device.Polylactic acid and polylactic-co-glycolic acid are used for erosion toallow an even rate of agent delivery.

The release of the agent from the compound is initially rapid, followedby a phase where the agent diffuses more slowly out of the compound andthen the remaining agent is released due to the degradation of thepolymer (FIG. 4). The extent of the initial release is controlled byplasticizers. High burst release is minimized by hydrophobicplasticizers; the opposite effect is achieved by hydrophilicplasticizers, which leach out of the polymer. The release of the agentdepends on the polymer molecular weight parameters. Low molecular weightpolymers or oligomers are preferred. Star-like copolymers ofhydroxyacids with polyhydric alcohols, such as pentaerythritol,mannitol, glucose, polyvinyl alcohol and others are also used becausethe branched carriers dictate the agent release by the degree ofbranching.

In an embodiment, the coating compound containing an agent comprises agel, liquid or powder coating that may contain an anti-fungal,anti-viral, antibiotic, anti-microbial, anti-parasitic, anti-protozoan,pH normalizing or acidifying (i.e. lactic acid), oxygen releasing agent(i.e. hydrogen peroxide), electromagnetic, alone or in some combination.Agents are further selected from the group LAE (lauramine arginine ethylester is a detergent-like agent and safe for food use), polylysine,stainless steel (confluent sleeve or as particles impregnated into theshell), steroidal, non-steroidal, hormonal, anti-oxidant,anti-inflammatory, antiseptic, or probiotic, alone or in combination.The device keeps the agent pressed against and penetrating the tissuewalls within the body cavity, making the combination of a cooling devicewith an agent possibly more efficacious (than either could performalone), with less spillage onto the outside perineum where it would beless effective or potentially caustic to the skin. Because the deviceretains the coating compound (agent) inside of the body cavity, lessconcentrated dosages and shorter duration of exposure to these compoundswill prove beneficial in reducing side effects and toxicities andpotentially improving efficacy. The coating compound may be for thepurpose of lubrication or ease of insertion alone or in addition to theabove listed purposes.

In an embodiment shown in FIG. 2, an optional removal means comprises anattachment 31. The attachment is an extension of the plug or anextension of the shell that is graspable such as a cord, bulb, stalk,button, loop attached to the plug or as an extension of the shell. In anembodiment, the attachment is a loop formed from cotton or suitablebiocompatible material, including the same material as the shell.

In an embodiment, the plug 13 is screwed into threads or snapped on atthe opening of the chamber after the filler is inserted, then sealedcompletely. The device further comprises a storage container (notshown). The device is packaged in according to GMP and FDA/CE markingrequirements and may or may not be sterile as in many other class I and11 devices for intravaginal use, such as tampons. There will be detailedinstructions/explanation of method of use, and safety guidelines.

The silicone or medical grade thermoplastic polymer shell is constructedin various forms, shapes, or sizes to accommodate varying body cavityshapes and sizes. In an embodiment, the shell is of similar fashion tothe current silicone breast implants or made of material currently usedin pessaries, vaginal dilators, or menstrual cups. A Stainless Steel(SS) sleeve may be used alone or in combination, and it may represent aconfluent surface or separate particles impregnated into the material ofthe shell. In an embodiment, the shell is more rigid, similar tosilicone used in re-useable ice cubes which are either filled withdistilled water, saline, or semi-solid hydrogel material. The hollowchamber of the device is filled with saline, distilled water, gas,hydrogel gel or other similar material that can either be frozen orcooled sufficiently (possibly through the mixing of two substancescreating an endothermic reaction) to effect a cure of the condition itis designed to treat/cure/or improve. The silicone, thermoplasticpolymer, or other suitable material (with or without stainless steelparticles or SS sleeve) is shaped to accommodate the shell of the deviceand may be a cylinder, a tube, oblong in shape, egg-shaped or anyvariety of configurations with varying lengths, widths, diameters, andcircumferences. Visualize a tampon for instance, a bullet, or a hotdog.Also, some of the devices will be shaped with an additionalcomponent-visualize a pacifier-to cool the external region of the bodycavity. The coating compound or SS may be applied to any part of thedevice.

The size of the invention (device) will be determined by the optimalsize to produce the safest, fastest, and most comfortable cure. Theremay be multiple sizes for different sizes and shapes of body cavities. Aremoval material may be a string of approximately 1-6 inches andattached to one end of the shell. In a reusable embodiment, the removalmaterial is washable. In a disposable embodiment, the string is anyinexpensive method of retrieving the device from the body cavity orpositioning the device within the cavity, and may be composed of but notlimited to, paper, cloth, plastic, or cotton string/cord and the like.In the case of a reusable device, the coating compounds are packaged inseparate containers or pouches with directions for mixing andapplication.

In an embodiment, the invention comprises a shell, with or without acoating compound, a freezable or cooling filler inside the shell'schamber, and the ability to remove and store the device, and an optionalstainless steel sleeve to cover the shell. Additionalmechanism/materials are optional for the purpose of inserting the deviceor accomplishing the intended goals of the invention. The cooling orfrozen filler substance can be liquid, gaseous, semisolid or solid, orgel. Additional materials can be used and modifications may be made tothe device depending on cost, safety, biocompatibility, effectiveness,and other medical considerations.

The silicone or other suitable material used to construct the shell'sshape is formed into a cylinder, tampon shaped, bullet shaped, or hotdogshape, rounded on one or both ends, or may be completely round,malleable, or changeable, but the size and shape are largely irrelevantas long the device accomplishes the intended purpose of the invention asdefined in earlier passages and as stated in the Claims Section. Theshell has a hollow internal chamber that contains the filler that can befrozen, made cold via the chemical reaction by mixing of two or moresubstances, or in the case of gas, may be inert. In an embodiment, thefreezable substance is saline or an hydrogel, although any othersuitable material or substance may be used that permits cooling or heattransference in body cavity tissues. The optional removal means can becleaned or sanitizable by routine household means in the case forreusable devices. In an embodiment, the removal means is a string. In anembodiment, the removal means is a ring or extension of flexiblematerial attached to one end of the shell. The stainless steel sleevewill be made in one or more sections so as to cover the shell partiallyor completely. SS particles may be impregnated into the shell materialto improve heat conductance and cooling capacity.

Saline, water, or hydrogels, (such as CMC gels used in breast implantssince 1994) are used as filler for the shell because of the inertproperties and proven safety when used inside the body. Inmanufacturing, the filler is added to the chamber in the shell and theshell is sealed, either as a single or multiple piece device.

As used herein, the words filler includes any substance or material thatcan be rendered frozen or sufficiently cold to accomplish the goal andnovelty of the invention. In an embodiment, the filler is a non-toxicpolymer gel, such as a medical grade gel refrigerant or biodegradablehydrogel. Saline is distilled water with dissolved sodium chloride(NaCl) or salt. The internal component of the device must be able tomade cold, cooler, or frozen when removed from a freezer or a coolingapparatus, or when two or more materials mixed together to produce anendothermic reaction, rendering the device cool or cold enough to beable to conduct heat away from warmer adjacent body cavity tissue viaheat absorption. Saline or analogous/similar substance/material (whenfrozen or cold) is the crux of the design of this device, and is not tobe in direct contact with the vaginal tissue. In the case of a SSsleeve, the temperature of the SS sleeve may be room temperature, priorto placement over the shell of the cooling device in a sequential mannerof use. The frozen or cooled saline, water, or hydrogel is the reasonthat this device is effective and the silicone (or shell material) isfirm enough to be placed in the vagina. Materials such as silicone andthermoplastic polymers do not freeze when subjected to the temperaturesin the common freezer. However, if the shell is impregnated withstainless steel particles, then the device may be placed into therefrigerator, or freezer, to cause the same cooling, heat absorbingcapacity. If the cooled or frozen device is too hard or stiff whenremoved from the freezer, a quick exposure to tap water will soften it.However, the device needs to be in a solid/semi-solid/frozen conditionor sufficiently firm in order for it to be inserted easily into the bodycavity. The device, once inserted will be slightly visible or palpableat the hymenal ring or introitus, or may be completely inserteddepending on the comfort level of theuser. The closer the device is tothe opening of the body cavity, the more heat absorption can be derivedfrom the external skin surface of the body cavity which helps cure boththe internal and external reservoir of pathogens. Deeper insertion ispossible or likely to occur as a result of the body cavity's musculatureand movement of the device's carrier. The vagina and vulvar tissue areinnervated differently, with less ‘sensation’ noted deep to the hymenalring within the vagina. However, it is within the vagina that the C.albicans and pathogens multiply best, and where biofilms stay intact.So, it is the length, width, diameter, circumference, and surface areaof the device that must be sufficient to accomplish cooling far enoughinto the vaginal cavity to effect a cure for the conditions describedherein. However, the device should not be adjacent to the cervix forcomfort reasons, unless it is determined by research that it is safe andefficacious to allow cervical contact. Cramping might occur if thedevice came into contact with the cervix due to its innervation. In anembodiment, the fluid or filler is a cold pack comprised of a shellhaving several compartments separated by breakable barriers. Each of thecompartments contain an ingredient which, when mixed with anotheringredient(s), chemically react together to create a cooling effect(endothermic reaction). One or more of the ingredients may be a liquid,powder, a semi-solid, gas, or solid substance. For appropriate use, thebarriers are broken allowing the contents to be mixed together.

Alternatively, the filler (saline, water or hydrogel) is an encasedinstant cold gel pack comprised of ingredients in breakablecompartments, that, when mixed together, form a cooling gel. Theaddition of the gel allows for later use. For re-use, the device isplace in a freezer, refrigerator, or cooling apparatus until needed.

The device has no active energy source. The human body temperature ofthe body cavity provides the warmth or heat to thaw the cooling orfrozen filler, thereby slowly cooling the adjacent tissues. The deviceacts to inhibit ‘filamentous’ yeast and bacterial overgrowth in bodycavities by passive heat absorption (thermal conductance) from the bodycavity or vaginal tissue (which is typically about 37° C. or 99° F.)through the shell walls of the device to slowly melt the cooled orfrozen filler gel, substance, etc. During treatment (about 45 minutes),the temperature of the tissues of the body cavity near the inserteddevice drop to approximately 68°-72 F within the first 10 minutes, thenslowly rises to approximately 78-80° F. by about 30 minutes,approximately 91-94° F. by about 45 minutes, and about 98° F. at about55 minutes as the filler thaws from heat absorption or thermalconductance. The treatment time and degree of tissue cooling (to bedetermined) is sufficient to force yeast into a dormant yeast ‘bud’morphology and out of the ‘filamentous’ virulent morphology, and toreduce bacterial multiplication and biofilm persistence found inconditions known as VulvoVaginal Candidiasis (VVC) or BacterialVaginosis (BV). Different duration of contact and amount of thermalenergy transfer may be used for altering the temperature inside the bodycavity, depending on the desired clinical effects. These temperaturesare used as an example of reducing the ‘filamentous’ forms of yeast(primarily C. albicans) causing it to morph back to the yeast ‘bud’morphology which is the dormant, non-disease causing state, and thusdoes not result in the release of the toxic cell contents which occurswhen anti-fungal chemicals destroy yeast cell walls. However, in thecase that research determines that the invention's cooling propertiesdoes result in Candida wall disruption, then the mechanism of action issimilar to chemical anti-fungal therapies, but without the delayed onsetof action and side effects of chemicals. Furthermore, anti-fungalchemicals contribute to the formation of biofilms whereas thisinvention's cooling device reduces biofilm formation and persistence,without adding chemicals, unless a coating compound is added. Thecoating compounds, for the purpose of this invention, will benon-biofilm producing. Biofilms disintegrate at a temperature less than80° F. The device will render the tissue cooler than this temperaturefor approximately 30 minutes, or longer depending on final volume andproperties of the selected filler, and desired clinical outcomes orefficacy which future research studies will elucidate.

The device is effective because it renders the surrounding tissuecooler, and thus the temperature sensitive pathogens are unable toreplicate/reproduce/persist in a biofilm that cause the disease orconditions. The inhibition, blocking or reversing of filamentous yeastmorphology and disrupting biofilms caused by both yeast and pathogenic(anaerobic bacteria) allows the body cavities' normal flora(lactobacilli) to reestablish and suppress the yeast and anaerobicbacteria by putting them into a dormant, non-invasive, ornon-filamentous form as when the temperature in the body cavity returnsto normal. The yeast and bacteria remain in either normal ‘physiologic’levels or dormant morphologies in non-diseased states (non-biofilms)unless conditions arise to initiate another growth cycle such as withanother temperature elevation, pH changes, douching, antibiotic use, orexposure to douches or sexual events. The addition of SS particles orstainless steel sleeve covering the device's shell helps the biofilm todissolve as a result of prolonged temperature reduction of thesurrounding tissue.

The cooling effect will also ameliorate the disturbing symptoms of yeastvaginitis (or other similar conditions, including BV). Itching, burning,and swelling respond to coldness by causing the capillaries to shrink(constrict) thus preventing the egress of a fluid into the tissue whichresults in swelling and pain. Coldness/ice packs are effective ways toreduce swelling caused by inflammation. With regard to the chamber beingfilled with a substance, the saline/hydrogel, or distilled water willremain in the solid state longer (absorb heat slowly) than tap water andwill freeze more quickly that water. Also, saline, hydrogel, anddistilled water are sterile and safe.

The process would be similar to the production of hydrogel or salinefilled silicone breast implants and/or re-useable ice cubes (made of asilicone shell or thermoplastic polymer), with the addition of aflexible string-loop-like material for easy removal of the device. Ifthe device is not to be a reusable device, then the string could be thesame as a tampon (i.e. cotton).

Other substances can be used to fabricate this device such as using atampon-like material (fiber) natural or synthetic, but not comprising anenclosure (shell). However, any absorbent material will result inmoisture being introduced and wetness will make this unsuitable for mostwomen (besides aggravating the condition it is designed to treat),including latex or other non-medical grade rubber or aromatic plasticmaterials, due to the possibility of allergic reactions. The sleeve willbe constructed of a medical grade stainless steel.

The user would keep the device(s) in a frozen or cooler state untilneeded, unless the mechanism of cooling is via the mixing of roomtemperature materials. The SS sleeve will be kept at room temperature,but if the SS particles are impregnated into the shell of the device,then it will be placed into a freezer, refrigerator, or coolingapparatus. Then, the device would be removed from the storage containerand placed under tap water for 3-5 seconds or less, then inserted intothe body cavity (vagina or analogous body cavity). The rounded end isinserted first, so that the removal means (if present) is closest to thebody cavity opening. The device may be adjusted using the attachment orthe removal means for comfort. The device remains in the body cavity forless than about 1 hour or until no additional cooling sensation isnoticed or experienced, and is then removed. A subsequent new orre-usable frozen or cooling device would be inserted at that time, ifneeded. This could be continued until the user was not experiencing anyitching or discomfort, possibly requiring repetitive episodes oftreatment every 4-6 hours, depending on the severity of the symptoms andlength of time needed to cure the infection. Future research willdetermine the optimal treatment duration, level of tissue coolingneeded, and frequency of treatment to cure VVC and BV. Once there-usable device has been used, it is removed from the body cavity,rinsed in warm, soapy water, dried and replaced back in the storagecontainer and placed back into the freezer or disposed of but, not byflushing. The SS sleeve may be reusable after being washed and dried.

The invention treats a condition called VVC caused by yeast (allgenus/species or yeast, specifically, Candida albicans,Candida-tropicalis, and Candida glabrata) and may be effective againstother microbes, such as protozoa, viruses, parasites, and bacteria,especially BV associated pathogens and biofilms. It stops the virulentgrowth and dissolves biofilms via cooling of the body cavity.

The addition of the coating compound may increase the efficacy of thecooling device while the cooling inhibits growth of the infectiouspathogen, and disrupts biofilms. The device achieves results by loweringthe temperature in the body cavity thus limiting the ‘filamentous’growth of yeast, pathogenic bacterial growth, and other microbes, whiledissolving biofilms. The agent in the coating compound adds anti-fungal,anti-viral, antibiotic, anti-microbial, anti-parasitic, anti-protozoan,pH normalizing or acidifying capabilities, steroidal, non-steroidal,hormonal, anti-inflammatory, anti-oxidant electromagnetically charged,anti-septic, oxygen generating, or probiotics, either alone or incombination to the cooling, tissue temperature reducing properties ofthe device.

The present invention also ameliorates the concomitant symptoms such asburning, itching, swelling, and pain associated with both BV, VVC,vaginal atrophy, radiation induced vaginitis, and dyspareunia frommultiple causes. However, this is not considered a novel or uniqueaspect of the invention since all cooling packs ameliorate inflammatoryconditions, irritation, swelling (edema), itching (pruritis), burning,and pain via the analgesic affect.

The invention acts as an anti-fungal (fungistatic or fungicidal) devicevia cold (heat absorbing) technology introduced by the device (shell andfiller), possibly by acting as an HSP 90 inhibitor (heat shockinhibitor) or cold shock inducer and acts to reverse yeast morphology,thus inhibiting any temperature sensitive component of a pathogen or thepathogen (in its entirety) by lowering the temperature of the bodycavity tissue which reduces filamentous growth of Candida species,halting and curing VVC outright. VVC and BV form biofilms, making theconditions more difficult to treat, thus the device's cooling effect,along with the optional application of an agent in a coating compoundsuch as those listed above, will provide additional mechanisms toeradicate infectious conditions in body cavities. The present inventiondisrupts the adjacent biofilm and modifies the body cavity temperatureto that below about 30° C., to approximately 20-28° C. for about 30minutes or longer, to allow penetration of hydrogen peroxide, lacticacid hydrogel, or some other agent to take effect. Biofilms disintegrateunder temperatures of 30° C. and have 100 fold fewer bacterial coloniesat these lowered temperatures, thus showing that some bacterial growthis temperature dependent. [0065]

Increasing the thermal conductance and cooling capacity via the additionof a SS sleeve or SS particles impregnated into the shell of the devicecreate an effective BV or VVC biofilm dissolving function, and prolongsthe duration of cooling. The introduction of a cooling, temperaturereducing device to a body cavity which cures infections is novel to theindustry. The use of a temperature reducing (heat transferring)intra-vaginal device for the purpose of inhibiting the filamentous,disease form of Candida yeast species, forcing the yeast to live intheir dormant (non-VVC), ovoid, yeast form is novel to the industry. Thecombination of adding a compound coating, to one or more of the abovemechanisms, is novel to the industry since no drug delivery devices usecooling as a component of their curative or therapeutic benefit. Nodevice serves as a substance retention device with a cooling componentfor the purposes described herein.

In an embodiment, an optional SS sleeve is added to the device todisrupt a pathogen's biofilm more quickly and effectively via thecooling effect (tissue temperature reduction). In addition, a biofilmmay be disrupted simply by the mechanical separation of the body cavitywalls via insertion of a device.

The following Example 1 is provided.

INTRODUCTION

An office based proof of concept study was conducted by Dr. KimberlyCull M.D. in 2009 with 20 of her patients who agreed to test thehypothesis that an intra-vaginal cooling device could relieve thesymptoms associated with yeast vaginitis such as itching, burning,swelling, and pain, and reduce the need for anti-fungal medicines.Scientific evidence is substantial in showing the analgesic effect ofapplying cold compresses to inflamed, bruised, or swollen tissue. Themechanism of action is that of vasoconstriction which reduces the amountof exudate (fluid) egress into damaged tissue. Furthermore, it is wellaccepted that warmth often caused by tight fitting clothing and moistureare needed for the proliferation of yeast. In fact, it is the loss ofnutrients, possibly as a result of lactobacillus (the ‘good’ bacteria)destruction from antibiotic exposure (lowering pH) that can cause yeastto ‘morph’ into their more virulent ‘filamentous’ form and becomeinvasive, resulting in the infection known as yeast vaginitis, even atnormal body temperatures. By altering one of the conditions necessaryfor yeast growth (temperature) with an intra-vaginal cooling device, itis hypothesized that yeast replication can be reduced, suppressing thisinvasive growth of yeast that causes the destruction of tissue,resulting in inflammation, pH changes, exudate and swelling. Theadjacent tissue's ambient temperature was modified by introducing acooling device into the vagina.

Background

Yeast vaginitis is a result of an overgrowth of Candida albicans,Candida torulopsis, Candida glabarata, or rarely, Candida Krusei, aloneor in some combination. This study proposed that modifying one of theambient conditions could prevent a full blown infection or treat thecondition outright. The ambient conditions that predispose to yeastvaginitis are heat, moisture, immune compromise, antibiotic destructionof favorable micro-flora such as lactobacilli, trauma, pregnancy,estrogen deficiency and uncontrolled diabetes among others.

Yeast cannot grow well under ‘cooler’ conditions, among other conditionsalready mentioned. Laboratory guidelines for culturing yeast uses atemperature of 36° C. as the lowest minimum temperature to encouragegrowth. A useful analogy would be that of making bread from flour, waterand yeast cultures. Yeast cultures are dissolved in warm water (95° F.)and the dough kept warm, otherwise, there will be no yeastmultiplication, which is essential for the production of carbon dioxidegas which makes the dough rise. With that principle in mind, yeastvaginitis can be ameliorated by keeping the surrounding tissue cooler,reducing the ability of yeast to multiply into an ‘overgrowth’ situationwhich causes the inflammation resulting in the symptoms of itching,burning, swelling, discharge and pain.

The study is a compelling example of the possibility that yeastvaginitis can be prevented, treated and even cured without theintroduction of chemicals. Furthermore, it is logical to assume that allspecies of yeast (four species are known to inhabit the vagina) areaffected by temperature, while most oral and OTC antifungal creams onlytreat one species of yeast.

Materials and Methods

Twenty patients were recruited for participation. Each presented forevaluation with the symptoms of itching, vaginal discharge, pain orburning of the vagina. Ages ranged from 18-45, and all were experiencingnormal monthly menstrual cycles. Participants were given a questionnaireto complete pre and post treatment. Histories and physicals ruled outconfounding conditions, such as sexually transmitted diseases (STD's),bacterial vaginosis, atrophic vaginitis and urinary tract infections(UTI's). Tests such as screening for gonorrhea and chlamydia, nitrazinetest, microscopic exam of the vaginal discharge to determine thepresence of hyphae or clue cells, were perfomed in all cases. Aurinalysis (UA) dipstick was used to rule out cystitis or pregnancy, aswell. All participants were current with pap smears. No trichomonas,bacterial vaginosis, UTI's, pregnancy, or STD's were found. Cultures foryeast returned positive in 15 of the 20 participants. All of theparticipants responded to a questionnaire (pre and post treatment).

The Treatment Device (TD) used was a frozen tampon. Each participantplaced the TD intra-vaginally. After one hour, or sooner, if noadditional cooling sensation was noted, participants removed anddiscarded the TD. If desired, they were could repeat the aboveinstructions with a second TD.

Results

Of the 20 women who agreed to participation in this study, all completedthe questionnaires, both pre and post treatment. Even though five of theparticipants failed to have a culture positive for yeast growth, theysufficiently qualified as suffering from yeast vaginitis based onsubjective questioning and other physical findings as identified onphysical exam. All participants answered positively to experiencingitching, discharge, burning, pain with sexual intercourse, or irritationand negatively to recent antibiotic use or antifungal use.

Eighteen participants thought that insertion of the TD was not painful,but some comments included that it was awkward and more painful, ifswelling was present. Two participants thought that insertion wassomewhat difficult, but they also had the most amount of swelling asnoted on physical exam. Wetness was experienced by all 20 participantsas a result of the melting of the TD.

All responses were positive as to relief of symptoms. Itching andburning were improved in all participants within 30 minutes ofinsertion, which continued after removal. Ten participants resumedsexual activity within 3 days after use of the TD; 6 within 5 days, and4 within 7 days.

All participants responded that they their condition improved or helpedafter the therapy. Sixteen thought they were completely cured of yeastvaginitis, while four were not sure if they were completely cured, butstill reported much improved symptoms. None resorted to additionaltherapies or medicines. All of the participants answered affirmativelythat they would consider this mode of therapy in the future, but wouldprefer not to have dampness as a side effect.

CONCLUSIONS

Ambient conditions in the vagina are tantamount to yeast proliferationand can be reduced via the alteration of certain ambient conditions.Disease or infections need certain conditions, substrates, nutrients,temperatures, predisposing factors to manifest and cause harm. While wecannot always prevent exposure to harm, we can easily alter oneessential ‘factor’ for disease progression.

The following is Example 2 is provided.

Reversing Yeast Morphology with a Cooling Device

Controlling Candida albicans via Physical Inhibition of HSP 90 Function.

Author: Kimberly Cull, M.D.

Background

VulvoVaginal Candidiasis (VVC) is the second most common form ofinfectious vaginitis, accounting for 30-35% of all cases, whileBacterial Vaginosis (BV) accounts for the diagnosis in 40-45% of womenwho present with vaginal complaints. VVC symptoms include vulvar andvaginal itching, burning, swelling, erythema, and pain, with or withoutdischarge, and with a normal acidic pH of 3.5-4.5. On the other hand, BVis diagnosed based on the finding of normal flora in the presence ofclue cells, elevated pH, a malodorous discharge, and relative lack of aninflammatory response. Biofilms appear to be a component of many formsof pathological ‘vaginitis-like’ conditions. Other types of vaginitisinclude aerobic vaginitis, desquamative inflammatory vaginitis, purulentvaginitis, and streptococcal vaginitis [33], among other noninfectiouscauses of vaginitis. Vaginal atrophy from estrogen deficiency andradiation induced vaginitis are two non-infectious forms of vaginitis.

Candida albicans accounts for 85-90% of all cases of VVC [24]. Otherspecies of Candida that can cause vulvovaginitis (VVC) include C.tropicalis, C. glabrata, C. krusei, among others. Risk factors for thedevelopment of VVC include excess heat, moisture, antibiotic destructionof favorable microflora such as lactobacillus, diabetes,immune-compromise, pregnancy, and oral contraceptive use [24]. The riskof disseminated candidiasis in cases of immune-compromise underscoresthe need for non-toxic, fast, and preventative means for treating thisvexing and recurring condition, for which current therapies areeffective only 70-80% of the time. These prescription andover-the-counter (OTC) topical anti-fungal medications result insignificant toxicities from yeast cell wall destruction with the releaseof enzymes, inflammatory immune responses, and allergic skin reactionsfrom caustic ingredients in the topical medications. This causes delaysin symptom relief and subsequent cure, potential interactions withsystemic medications, and the risk of anti-fungal drug resistance due torepetitive or inaccurate therapies.

The focus of this scientific review is to show how Candida HSP (heatshock protein), specifically HSP 90, is the target for treating both theetiology and symptom associated with VVC, primarily by reducing the heatshock response or invoking a cold shock response, thus reversing themorphological switching that plays the fundamental role in yeastvirulence. ‘Heat shock protein's (HSP) are molecular chaperones whoseproduction is increased as a result of environmental stressors such aselevated temperature and ‘oxidative’ inflammation. This review willaddress the link between HSP 90, yeast morphological states, signaltransduction pathways, tissue destruction, and excess heat in bodycavities. Furthermore, the author will illustrate a molecular mechanismof action explaining why an intra-vaginal cooling device amelioratessymptoms immediately and reverses yeast morphological pathobiology,curing the condition known as VVC, without medication or chemicals.

Morphological States of C. albicans and Molecular Pathways

C. albicans exists in three morphological states, yeast, psuedohyphae,and hyphae. The latter two, collectively considered ‘filamentous’,account for Candida's virulence, and are the two forms responsible forVVC, among other human fungal infections. C albicans' morphologies areenormously plastic, able to change readily from one form to another [6].Depending on environmental conditions such as temperature, nutrientdepletion, pH or CO2 elevation, the yeast can change shape in order tosurvive hostile or changing environments. The requirement fortemperature elevation to induce morphogenesis has remained enigmatic[1]. In vivo, all three morphologies might coexist at a single site ofinfection [2]. This phenotypic switching clearly plays a role invirulence [8]. At higher temperature and pH, the hyphal formpredominates [9]. At 37 C and with exposure to serum, unbudded yeastconverts to hyphal forms, whereas at 35 C and neutral pH, bothpseudohyphal and hyphal forms predominate. At 30 C (86 F), and a pH 4.0or less, dormant, yeast forms exist [10].

The yeast-to-hyphal or bud-to-hyphal transition (BHT) is triggered by awide range of environmental factors such as carbohydrates, amino acids,salts, pH changes, temperature increases (or decreases), nutrientdepletion, growth within a matrix (biofilms)[38]. In C. albicans, thisBHT is a result of signal transduction pathways [39]. The two moststudied of these are the mitogen-activated protein kinase (MAPK) whichregulates cell wall integrity as a result of environmental perturbations(stress) and the cAMP-protein kinase A (PKA) pathway which is thenutrient sensing pathway or the fermentable growth medium’ (FGM) pathway[40] and possibly a temperature integrating pathway via the Ras 1 cAMPPKA [1,43].

The other pathways include an amino acid sensing, ammonium sensing,mechanosensing, and the Tup1-Nrg1 mediated pathway that REPRESSESfilamentation independent of the cAMP-PKA (EFg1) or MAPK (Cph1) pathways[41,42]. Nrg 1 is a DNA binding protein that represses filamentousgrowth in C. albicans, probably acting through co-repressor Tup 1[62].Braun et al. (2001) showed that growth in serum at 37 C resulted in areduction in Nrg 1 mRNA, suggesting that filamentous growth is inducedby down-regulation of Nrg 1[62], making it possible that loweringtemperature causes an up-regulation of Nrg 1 which repressesfilamentation. The Tup1-Nrg 1, MAPK, mechanosensing pathways warrantcloser examination in relation to the ‘physical’ temperature conditionswhich impact C. albicans' morphologic switching, in addition to thecAMP-PKA pathway's potential dual role in both nutrient and temperaturesensing.

To date, there have been identified 16 out of 480 molecules tested whichinhibit the cellular signaling that block the BHT. These inhibitorsaffect protein kinases, protein phosphatases, Ras signaling pathway(nutrient sensing), G-protein coupled receptors (GPCR), calciumhomeostasis, nitric oxide, and guanylate cyclase [41]. The temperatureeffect on GPCR is receptor specific, as in many mammalian receptors, adecrease in temperature results in up to a six fold increase in receptorexpression [44]. The explanation that protein production is slowed(receptors down-regulated) by cooling is thought to be dependent uponnot overloading the translocation machinery, protein processing, orintracellular trafficking. Thus, lowering temperatures could reduceproteolytic activities [45] or up-regulate cold shock proteins such aschaperones [44]. The focus of this research proposal is to learn moreabout the link between physical ‘temperature’ reduction as it relates toreversing yeast morphology, inhibiting or blocking yeast filamentationand how the BHT is dynamic and reversible, which allows C. albicans theability to survive and thrive in many host conditions and substrates.

Environmental Sensing in Fungi

Several types of environmental sensing responses in fungi have beenidentified. They include responses from low oxygen, nutrientdeprivation, osmotic stress, and contact sensing. Contact sensing infungi regulates differentiation which is crucial for virulence [54]. C.albicans, contact sensing results in the formation of invasive growth[46]. This mechano-sensing involves mechanosensitive (MS) ion channels,G-protein couple receptors (GPCR), and integrins. These MS ion channelsopen in response to physical stimuli that affect the membrane [47,48]. Abiologic result occurs as a result of a mechanical stimulus [42]. C.albicans exhibit a behavior termed thigmotropism which result in thereorientation of hyphae in response to ridges and are believed to be aresult of MS channel function [55]. This thigmotropism might guideinvading hyphae towards gaps between cells, facilitating the tissuepenetration necessary for virulence[42] Hence, C. albicans cells sensethe properties of a surface and produce characteristic biologicalresponses [42].

GPCR are also implicated in mechanosensing. They are activated by lightabsorption, ligand binding, and mechanical forces described asmechanical stretching [58], shear stress [57], hypotonic stress, andmembrane fluidity [42]. For instance, if a mutant C. albicans lack theGPCR Gpr1p, filamentation in response to contact is defective whereastheir response to other environmental cues is normal [49-51]. Thesebud-to-hyphal (BHT) defects were stronger on solid hyphal inducing mediacompared to liquid media. [39]. This supports the observation thathyphal forms are more capable of solid tissue (vagina) penetration,whereas the yeast bud forms are more suited for dissemination throughthe bloodstream. Likewise, on solid surfaces, C. albicans can formbiofilms [52]. Overall, little direct investigation of how mechanicalforces influence GPCR function has been performed [42].

Integrins are transmembrane signaling proteins that respond to forcesexerted perpendicular to the surface of a cell membrane [59]. Usingatomic force microscopy, the fungal cell wall undergoes an oscillatingmotion whose frequency is temperature-dependent and results in abiologic activity [60]. Thus, temperature changes create mechanicalperturbations that change the vibrational energy of the cell membranes,triggering integrins, GPCR's, or MS ion channels to perform theirbiological functions. A useful ‘physics’ analogy would be that of coldcausing contraction of the molecules of gas, liquid or solid materials,whereas heat causes the expansion of these same physical states ofmatter. Regardless, fungi have evolved to adapt to many environmentalconditions of which temperature reigns as important candidate formanipulation.

Heat Shock Proteins as Molecular Controllers of Morphology

Heat Shock Proteins (HSP's) are the most abundant proteins in microbesand humans, and act as molecular chaperones, serving both housekeepingfunctions and adaptation for survival in hostile conditions, such assurviving the mammalian host's immune system. Yeast HSP 90 is essentialfor the viability of yeast [12]. As a molecular chaperone, HSP 90ensures the correct conformational activity, localization, andproteolytic turnover of a range of proteins that are involved in threeprocesses: cell growth, differentiation, and survival [27,28,29]. In2004, Albarrag opined that HSP 90 may play role in the pathogenesis andvirulence of C. albicans [11]. In fact, HSP 90 is the key mediator of C.albicans' pathobiology, controlling nearly all facets includingmorphogenesis, biofilm formation, virulence, and anti-fungal drugresistance [20] [21]. In non-stressed cells, HSPs are present in lowconcentrations; but when stressed, they accumulate to high levels [4].According to Bergman and Segal, HSP 90 suppresses phenotypic variationunder normal conditions, but releases this suppression of variation whenfunctionally compromised [25]. Thus, in conditions of elevatedtemperature, these proteins increase 5-6 fold, thus explaining the ‘heatshock response’ [3]. This leads to a switch from yeast to ‘filamentous’forms which are considered invasive and promote tissue penetration whichis explains most of the symptoms found in VVC [2].

Yeast HSP 90 ‘reversibly’ governs morphogenetic transition from yeast tofilamentous growth [1]. This transition is regulated by environmentalcues such as exposure to serum, pH and CO2 elevation, nutrientlimitation, that is contingent upon elevated temperature to inducemorphogenesis [15] [16]. In other industries, an example of yeastactivity or growth as a function of temperature can be shown with thefermentation process of S. cerevisiae (84% homologous to C. albicans).Yeast cells are exposed to complex and varied carbon sources, and‘stressed’ by anaerobic growth, high specific gravity, ethanol, andtemperature ‘shocks’. Whereas, during times of storage for extendedperiods of time, yeast is kept dormant at lowered temperatures. [3].

In 2009, Shapiro showed that by compromising yeast HSP 90 PRODUCTION,not FUNCTION via biochemical, pharmacologic, or genetic pathways, thetemperature and serum exposure requirement could be mitigated, causing ayeast to filamentous switch. [1]. Shapiro further demonstrated that inliquid rich media at 30 C (86 F), C. albicans exists as budding yeasts,and that even with exposure to serum, this temperature did not causefilamentation. She suggested that Yeast HSP 90 is essential formorphogenesis which makes targeting HSP 90 production the idealtherapeutic strategy [1]. In fact, inhibition of HSP 90 ‘function’ in C.albicans blocks the emergence and maintenance of resistance to azoles,enhancing their efficacy in vivo [7]. Thus, exposing C albicans to a‘cooling’ environment after a ‘heat shock’ resulted increased HSP 90production, means that reducing the temperature either impairs HSP 90function or that another pathway has become activated such as the ‘coldshock response’. Since temperature elevation raises HSP 90 productionvia up-regulation that occurs when cells are heat shocked from 25 C to37 C for 30 minutes [34], inhibiting HSP 90 function via tissuetemperature reduction for 30-40 minutes should reverse the ‘filamentous’forms back to the non-disease causing ‘yeast’ morphologies, since thismorphological process is reversible. The answer or conclusions awaitfurther study.

More confirmation of this temperature ‘essentiality’ was shown inanother study conducted by Shapiro in 2012, an HSP-90 co-chaperone(Sgt1) was identified that also coupled temperature sensing withmorphogenesis. It was shown that by inhibiting HSP 90 function, and to alesser extent, Sgt1 function, the Candida susceptibility to azoles andechinocardins was enhanced, creating a fungicidal, rather thanfungistatic combination of these anti-fungal drugs, and reduce biofilmresistance at 30 C [5]. By reducing the temperature to 30 C, Sgt1 andHSP 90 activities were reduced, verifying that the virulence of C.albicans is dependent on temperature elevation, making the author'stheory that virulence is diminished with temperature reduction.Likewise, biofilms begin to form at 30 C and above and start todisintegrate at temperatures below 30 C and under conditions of lessenergy and diminishing nutrients [23]. Thus, temperature reduction viaan intra-vaginal cooling device could have a potential role in BV andaerobic vaginitis since both are thought to be resistant to therapy as aresult of their protective biofilms which are adherent colonies ofmicroorganisms, heldtogether by a matrix of polysaccharides, proteins,and nucleic acids and which have a predilection for skin and mucosalsurfaces [23].

Thus, lowering tissue temperature acts as an HSP 90 inhibitor or blockerby reducing filamentous growth, forcing or pushing it back to the‘yeast’ form which is the dormant, co-inhabitant state of Candidaspecies. This ‘reversibility of morphology’ feature is also why theaddition of heat can restart the virulent morphological cycle thatcauses VVC.

Combined Human HSP 70 and Yeast HSP 90 Explains VVC Symptom

Yeast HSP 90 is located on cell wall surfaces at differentconcentrations influenced by environmental and nutritional conditionswith large concentrations at the tips of the hyphae. The tips of thehyphae play a role in virulence since this is the site of enzymesecretion which results in tissue penetration, protein and lipiddestruction, and host responses, as a result [13]. Host responses in VVCinclude vaginal discharge, itching, burning, swelling, and pain. This isdue to a combination of the body's immune response to the enzymaticdestruction of tissue resulting in inflammation, which then results inhuman HSP 70 up-regulation from this oxidative stress. HSP 70 wasrecently recognized as one of the antimicrobial proteins present in thevagina [35, 36]. Inflammation occurs as a result of an activation ofvarious signaling molecules involving prostaglandin and arachidonic acidproduction as a result of Phospholipase A. Both of these further inducethe heat shock response leading to increased HSP synthesis [26]. Theinitial increase in heat triggers the yeast HSP 90 to ‘morph’ intofilamentous forms, which then results in inflammation which induces boththe host and the pathogen to respond with increased HSP productioncreating a dynamic, vicious cycle of growth and destruction.

Likewise, in 1998, it was discovered that HSP 90 and HSP 70 (Gene, 2006)cause the release of nitric oxide, a smooth muscles relaxer,vasodilator, and potent inhibitor of platelet aggregation [17]. Nitricoxide has an antimicrobial activity against a wide variety ofmicroorganisms [37]. This over-production of nitric oxide wasdemonstrated in the murine model of oral candidiasis [14]. HSP 90 alsoactivates the kinin-forming cascade leading to bradykinin release,causing edema and more vasodilation, and explains the notable swellingfound with VVC. By inhibiting HSP 90, the nitric oxide formation isdecreased with a resultant decrease in cell damage, exudative discharge,erythema, and swelling [18]. It has long been established that applyingcooling packs to reddened, swollen, inflamed, and sore tissue willlessen those symptoms, and now we have a plausible molecular explanationto accompany the well-known role of coolness in producingvasoconstriction, reducing fluid egress into tissue.

To summarize, both host cells and microbes are confronted with dramaticalterations in living conditions during an infection (i.e. vasodilationfrom nitric oxide, bradykinin release, phospholipase and proteinaseproduction, histamine release, hemolytic activity, adhesion to vaginalepithelial cells causing biofilms, inflammatory/immune cell response,edema, and erythema). ALL of these contribute to further alterations inpH, CO2 production, increased heat and inflammation. Thus, reducing thesurrounding tissue temperature can improve both the signs and symptoms(caused by the inflammation), as well as ‘curing’ VVC by pushing‘filamentous’ Candida growth back to the dormant ‘yeast’ forms or byblocking the continual ‘filamentous’ transformation, allowing the hostimmunity to repair and heal the traumatizedtissue. Thus, by lowering thetemperature of the yeast environment, both human HSP 70 and Candida HSP90 are relegated to their ‘housekeeping’ rather than cytoprotective‘heat shock’ roles, caused by exposure to temperature elevations.

Immune Response to HSP 90

HSP's are major antigens and induce a very strong humoral and cellularimmune response [4]. Many of the cellular responses were mentionedpreviously. In mycology, HSP 90 has been identified as a potentialtarget for immunotherapy, since the activation HSP 90 was inhibited by amonoclonal antibody to HSP 90 known as Mycograb [4]. Mycograb is a humanrecombinant protein that recognizes the middle domain of fungal HSP 90.When combined with the antifungal drug amphotericin B, it has been shownto reduce mortality in systemic candida with an 84% response rate formultiple species of yeast, including C. albicans, C. glabrata, and C.tropicalis [30]. This illustrates the presence of ‘heat shock proteins’in all or most species of pathogenic yeast and supports the author'stheory that all species of Candida that cause VVC will respond to acooling device, not just C. albicans.

Autoimmune or Chronic Disease Connection to HSP 90

“Provoked vestibulodynia, the most common form of vulvodynia(unexplained pain of the vulva), is a prevalent, idiopathic paindisorder associated with a history of recurrent candidiasis (VVC)” [22].In a subset of female mice subjected to recurrent Candida albicansinfections, they developed a mechanical allodynia (painfulhypersensitivity to touch) of the vulva. This subset also exhibitedhyperinnervation with peptidergic nociceptor and sympathetic fibers.Long lasting allodynia was also observed after a single, extendedCandida infection, as well as repeated vulvar inflammation induced witha mixture of fungal antigens [22]. Tolerance to self antigens may bedistorted by frequent encounters of the immune system with microbialantigens with high similarity to self [19]. Because Yeast HSP and humanHSP 90 share similarity in composition, and due to HSP wide distributionand their homology among different species, it is not surprising thatautoimmune phenomena can be explained “as when HSP expression and HSPimmune responses are regulated inappropriately.” [4] Thus, ongoinginflammatory processes, repetitive exposure to HSP and microbialantigens, with continued immune responses can explain why chronicdiseases such as vestibulitis or vulvodynia exist, and may represent anautoimmune phenomenon. It is due to the structural homology betweenmicrobial and ‘self’ HSP [4]. Preventing and reversing these antigenicexposures, without the need for chemicals found in antifungal therapies,is the basis for a vaginal cooling device as treatment for VVC.

CONCLUSION AND FUTURE CONSIDERATIONS

Being able to treat the etiology of VVC by changing yeast morphologiesand disrupting biofilms via temperature reduction with a fast,non-toxic, and simple device whose ‘cooling’ mechanism also relieves theconcomitant symptoms such as itching, burning, swelling, and pain oncontact, is the basis for this review of the literature. A ‘Proof ofConcept’ study was conducted by the author in 2009 on twenty patientseach suffering from an uncomplicated case of VVC. Each used a tap watersaturated, frozen tampon for less than two hours, and everyoneexperienced immediate relief, and none resorted to additional therapies.

The need exists to complete more detailed studies with the author'spatent pending polyurethane or silicone medical grade device, filledwith a freezable gel, to determine the appropriate degree of temperaturereduction, duration of use, and number of times (dosage) needed whichresults in a cure of VVC while simultaneously ameliorating the symptomsof VVC. With the availability of a rapid, sensitive immunochromatographytest (ICT) known as the CandiVagi assay (SR2B, Arville, France), thestudy will be able to discriminate between Candida carriage and Candidainfection, with the specificity to exclude any women with eitherbacterial or trichomonal vaginitis [31]. Yeast cultures can determinethe exact species and further validate the amount of growth or severityof Candida infection, which will be correlated with clinical exams,cervicovaginal lavage to measure host immune response as well asquantifying levels of lactobacilli, and other vaginal microflora viaPCR. Expression levels of a filament-specific transcriptional regulatorcan determine C. albicans morphology and virulence according to Carlisleet al [49]. Using Transmission Electron Microscopy by fixing C. albicansin the McDowell-Trump fixative, it will be possible to show themicrostructural and gross morphological changes in yeast cells, afterexposing yeast to varying temperatures [53]. HSP 90 levels and functionshould be quantified and qualified with regard to temperaturefluctuations.

Intravaginal sensors such as those used by Hill et al (2005) to measurevaginal oxygen and carbon dioxide levels during menses can be used.Physiologic levels are atmospheric in the vagina, with a slighttransient increase in oxygen levels with tampon insertion. Carbondioxide levels remained unchanged with tampon insertion [32]. TheseNeotrend sensors are fiber optic based units and less than 0.5 mm indiameter and 23 mm in length. These sensors record temperatures, pH,pCO2, pO2 levels continuously [32], elucidating the physiologic aspectsof cooling in a body cavity. Other parameters and diagnostic tools maybe considered as the data is collected and analyzed. Biofilm disruptionas a result of temperature reduction should be addressed, eitherseparately or in a combined microbiologic and clinical trial. Theexistence of reconstituted human vaginal epithelium (RHVE) used byPietrella et al [61] for evaluation of the vaginal immune response to C.albicans, may be useful in reconstructing biofilm formation.

Patient questionnaires after clinical evaluation and examinations areperformed will be used prior to device insertion, during the treatment,immediately after-wards, within 24 hours, at one week, and one monthlater. Results from the questionnaires, clinical assessments, and testresults will be correlated to determine the device's efficacy and helpelucidate a mechanism of action.

The foregoing descriptions of specific embodiments and examples of thepresent invention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteachings. It will be understood that the invention is intended to coveralternatives, modifications and equivalents. The embodiments were chosenand described in order to best explain the principles of the inventionand its practical application, to thereby enable others skilled in theart to best utilize the invention and various embodiments with variousmodifications as are suited to the particular use contemplated. It istherefore to be understood that within the scope of the appended claims,the invention may be practiced otherwise than as specifically describedherein.

What is claimed is:
 1. A method of disrupting and inhibiting the growthof a pathogen in a vagina of a patient, comprising repeating, until atherapeutic effect is achieved, the steps of: providing a devicecomprising a cooling or freezable filler material; cooling or freezingthe device; inserting the cooled device into the vagina of the patient;allowing the device to remain in the vagina for a period of timesufficient to promote a condition in the pathogen selected from:dormancy, changed morphology, cessation of growth, disruption of abiofilm created by the pathogen, and any combination thereof; andremoving the device from the vagina, and; obtaining the therapeuticeffect after repeating the steps, wherein the therapeutic effectcomprises a drug-free cure of bacterial vaginosis vulvovaginalcandidiasis or both.
 2. The method of claim 1, wherein the pathogen is afungus.
 3. The method of claim 2, wherein the fungus is a yeast.
 4. Themethod of claim 3, wherein the yeast is genus Candida.
 5. The method ofclaim 1, wherein the pathogen is a bacterium.
 6. The method of claim 1,wherein the period of time is between 30 and 60 minutes.
 7. The methodof claim 1, wherein the steps are repeated at least two times.
 8. Themethod of claim 1, wherein the steps are repeated at least three times.9. The method of claim 1, wherein a temperature of vaginal tissue goesbelow 30° C. during each period of time.
 10. The method of claim 1,wherein a temperature of a portion of vaginal tissue goes below 26° C.during each period of time.
 11. The method of claim 1, wherein atemperature of a portion of vaginal tissue goes below 22° C. during eachperiod of time.